Peter D. East

Regulation of lepidopteran foregut movement by allatostatins and allatotropin from the frontal ganglion

The frontal ganglion and associated neuronal pathways in larvae of the noctuid moth Helicoverpa armigera have been studied immunocytochemically with antisera against the endogenous neuropeptides, the allatostatins (helicostatins), and allatotropin. Two pairs of large ganglionic neurones contain allatostatin immunoreactivity, with the anteriormost of these pairs showing colocalisation with allatotropin. Allatostatin and allatotropin axons exit the frontal ganglion in the recurrent nerve and traverse the surface of the crop to give terminal arborisations around the stomodeal valve. There is a greater degree of lateral branching of allatotropin axons compared with allatostatin axons over the crop musculature. In vitro experiments show that the two types of peptides have antagonistic effects on the spontaneous myoactivity of the crop musculature. Allatotropin is myostimulatory at concentrations as low as 10−16 M, enhancing both frequency and amplitude of peristaltic waves of contraction. All members of the helicostatin family inhibit peristalsis completely at concentrations of 10−7–10−6 M and, to varying degrees, at 10−10–10−8 M. On the basis of this evidence, it is suggested that peptidergic neurones of the frontal ganglion play a major part in regulating foregut motility through the antagonistic actions of the allatostatins and allatotropin. J. Comp. Neurol. 413:405–416, 1999.

Lepidopteran Peptides of the Allatostatin Superfamily

Peptides of the allatostatin superfamily with the C-terminal amino acid sequence -YXFGL-NH2 have been isolated and identified from the lepidopterans, the codling moth, Cydia pomonella (Tortricidae) and the bollworm, Helicoverpa armigera (Noctuidae). The peptides, designated cydiastatins and helicostatins respectively, were monitored during purification with radioimmunoassays based on the callatostatins of the blowfly Calliphora vomitoria. The eight peptides from each of the two species appear to form an homologous series with four identical and three that differ by a single amino acid. This study demonstrates the ubiquitous nature of this family of peptides in insects.

A peptidomics study reveals the impressive antimicrobial peptide arsenal of the wax moth Galleria mellonella

The complete antimicrobial peptide repertoire of Galleria mellonella was investigated for the first time by LC/MS. Combining data from separate trypsin, Glu-C and Asp-N digests of immune hemolymph allowed detection of 18 known or putative G. mellonella antimicrobial peptides or proteins, namely lysozyme, moricin-like peptides (5), cecropins (2), gloverin, Gm proline-rich peptide 1, Gm proline-rich peptide 2, Gm anionic peptide 1 (P1-like), Gm anionic peptide 2, galiomicin, gallerimycin, inducible serine protease inhibitor 2, 6tox and heliocin-like peptide. Six of these were previously known only as nucleotide sequences, so this study provides the first evidence for expression of these genes. LC/MS data also provided insight into the expression and processing of the antimicrobial Gm proline-rich peptide 1. The gene for this peptide was isolated and shown to be unique to moths and to have an unusually long precursor region (495 bp). The precursor region contained other proline-rich peptides and LC/MS data suggested that these were being specifically processed and were present in hemolymph at very high levels. This study shows that G. mellonella can concurrently release an impressive array of at least 18 known or putative antimicrobial peptides from 10 families to defend itself against invading microbes.

Proteomic analysis of the peritrophic matrix from the gut of the caterpillar, Helicoverpa armigera.

The peritrophic matrix from the midgut of the caterpillar, Helicovera armigera, was solubilized by treatment with anhydrous trifluoromethanesulfonic acid, apparently by depolymerisation of its chitin component. This allowed the efficient extraction of proteins in a technique that may be broadly applicable to the analysis of other structures containing chitin. Gel electrophoresis and mass spectrometry of tryptic peptides were used to identify the extracted proteins with gut-expressed cDNA sequences. The major proteins of this cohesive, digestion-resistant structure are chitin deacetylase-like and mucin-like proteins, the latter with multiple chitin-binding domains that may cross-link chitin fibrils to provide a barrier against abrasive food particles and parasites, one of the major functions of the matrix. Other proteins found in the H. armigera gut peritrophic matrix suggest that the matrix is a dynamic, complex structure that may participate in the immobilization of digestive enzymes, actively protect the gut from parasite invasion and intercept toxins such as lectins and Bacillus thuringiensis crystal proteins.

Diversity of aminopeptidases, derived from four lepidopteran gene duplications, and polycalins expressed in the midgut of Helicoverpa armigera: Identification of proteins binding the δ-endotoxin, Cry1Ac of Bacillus thuringiensis

Helicoverpa armigera midgut proteins that bind the Bacillus thuringiensis (Bt) delta-endotoxin Cry1Ac were purified by affinity chromatography. SDS-PAGE showed that several proteins were eluted with N-acetylgalactosamine and no further proteins were detected after elution with urea. Tandem mass spectral data for tryptic peptides initially indicated that the proteins resembled aminopeptidases (APNs) from other lepidopterans and cDNA sequences for seven APNs were isolated from H. armigera through a combination of cloning with primers derived from predicted peptide sequences and established EST libraries. Phylogenetic analysis showed lepidopteran APN genes in nine clades of which five were part of a lepidopteran-specific radiation. The Cry1Ac-binding proteins were then identified with four of the seven HaAPN genes. Three of those four APNs are likely orthologs of APNs characterised as Cry1Ac-binding proteins in other lepidopterans. The fourth Cry1Ac-binding APN has orthologs not previously identified as Cry1Ac-binding partners. The HaAPN genes were expressed predominantly in the midgut through larval development. Each showed consistent expression along the length of the midgut but five of the genes were expressed at levels about two orders of magnitude greater than the remaining two. The remaining mass spectral data identified sequences encoding polycalin proteins with multiple lipocalin-like domains. A polycalin has only been previously reported in another lepidopteran, Bombyx mori, but polycalins in both species are now linked with binding of Bt Cry toxins. This is the first report of hybrid, lipocalin-like domains in shorter polycalin sequences that are not present in the longest sequence. We propose that these hybrid domains are generated by alternative splicing of the mRNA.

Identification of the dipteran Leu-callatostatin peptide family: characterisation of the prohormone gene from Calliphora vomitoria and Lucilia cuprina.

The prohormone gene encoding the Leu-callatostatin peptides has been isolated from a Calliphora vomitoria genomic DNA library and its homologue was cloned from genomic and cDNA libraries of another blowfly species, Lucilia cuprina. Gene and prohormone structure and organisation are essentially identical in the two species. The Leu-callatostatin gene consists of at least 3 exons. The prohormone is encoded on exons two and three and the two blocks of putative Leu-callatostatin peptides are carried on separate exons. It is 180 amino-acids long, begins with a short signal peptide and contains two blocks of tandemly arranged Leu-callatostatin peptides separated by an acidic spacer region. The prohormone contains 5 copies of the C-terminal sequence -YX FGL characteristic of the Leu-callatostatin family. Complete endoproteolytic processing at all possible pairs of basic amino acids would generate 5 different Leu-callatostatin octapeptides. Two larger Leu-callatostatins could be released if processing was not complete at two of the sites. None of the 3 peptides encoded in the first block was identified in previous purification studies of the callatostatin peptides. The second block, located at the carboxyl end of the prohormone, contains two peptide sequences identical to the previously isolated Leu-callatostatins 1 and 4. The absence of independent copies of Leu-callatostatins 2 and 3 on the prohormone establishes that endoproteolytic cleavage of the precursor does not invariably proceed to completion and that Leu-callatostatin 2 must be derived by N-terminal processing of the parent peptide Leu-callatostatin 1. Reverse transcriptase PCR analysis of mRNA from brain and midgut, the two major sites of Leu-callatostatin expression, shows that the prohormone sequence at these two sites is identical, ruling out the possibility that different populations of peptides are expressed in these two tissues as a result of alternative RNA splicing.

The discovery and analysis of a diverged family of novel antifungal moricin-like peptides in the wax moth Galleria mellonella

Screening for components with antifungal activity in the hemolymph of immune-stimulated Galleria mellonella larvae led to the identification of four novel moricin-like peptides (A, B, C3 and D). Subsequently, eight moricin-like peptide genes (A, B, C1-5 and D) were isolated and shown to code for seven unique peptides (mature C4 and C5 are identical). These genes contained single introns which varied from 180 to 1090bp. The moricin-like peptides were particularly active against filamentous fungi, preventing the growth of Fusarium graminearum at 3 microg/ml, and were also active against yeasts, gram positive bacteria and gram negative bacteria. Searches of the databases identified 30 moricin-like peptide genes which code for 23 unique mature peptides, all belonging to the Lepidoptera (moths and butterflies). The first comprehensive phylogenetic analysis of the moricin-like peptides suggested that they fall into two basic classes which diverged a long time ago. The peptides have since diversified extensively through a high level of gene duplication within species, as seen in G. mellonella and Bombyx mori. The restriction of moricin-like peptides to the Lepidoptera combined with their potent antifungal activity suggests that this diverse peptide family may play a role in the defence response of moths and butterflies.

Localisation of sulfakinin neuronal pathways in the blowfly Calliphora vomitoria.

The distribution of neurones immunoreactive to antisera raised against the undecapeptide C-terminal fragment of drosulfakinin II (DrmSKII), Asp-Gln-Phe-Asp-Asp-Tyr(SO3H)-Gly-His-Met-Arg-Phe-NH2, has been studied in the blowfly Calliphora vomitoria. Antisera were preabsorbed with combinations of the parent antigen, the tetrapeptide Phe-Met-Arg-Phe-NH2 and cholecystokinin, the vertebrate sulfated octapeptide (CCK-8), Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2, in order to ensure specificity for the sulfakinin peptides of C. vomitoria (the nonapeptide callisulfakinin I is identical to drosulfakinin I and callisulfakinin II differs from DrmSK II only by the presence of -Glu3-Glu4- in place of -Asp3-Asp4-). Only four pairs of sulfakinin-immunoreactive neurones have been visualised in the entire nervous system. These occur in the brain: two pairs of cells situated medially in the caudo-dorsal region close to the roots of the ocellar nerve and two other pairs at the same level but positioned more laterally. Despite the small number of sulfakinin-immunoreactive cells, there are extensive projections to many areas of neuropile in the brain and the thoracic ganglion. The pathway of the medial sulfakinin cells extends into each of the three thoracic ganglia and a metameric arrangement of sulfakinin neuronal projections is also seen in the abdominal ganglia. Neither the dorsal neural sheath of the thoracic ganglion, nor the abdominal nerves contain sulfakinin-immunoreactive material. These observations suggest that the sulfakinins of the blowfly function as neurotransmitters or neuromodulators. They do not appear to have a direct role in gut physiology, as has been shown by in vitro bioassays for the sulfakinins of orthopterans and blattodeans. In addition to the neurones that display specific sulfakinin immunoreactivity, other cells within the brain and thoracic ganglion are immunoreactive to cholecystokinin/gastrin antisera. There are, therefore, at least two types of dipteran neuropeptides with amino acid sequences that are similar to the vertebrate molecules cholecystokinin and gastrin.

Identification of the dipteran Leu-callatostatin peptide family: the pattern of precursor processing revealed by isolation studies in Calliphora vomitoria.

Information from the Leu-callatostatin gene sequences of the blowflies Calliphora vomitoria and Lucilia cuprina was used to develop antisera specific for the variable post-tyrosyl amino-acid residues Ser, Ala and Asn of the common Leu-callatostatin C-terminal pentapeptide sequence -YXFGL-NH2. Radioimmunoassays based on these antisera were used to purify peptides from an extract of 40000 blowfly heads. Five neuropeptides of the Leu-callatostatin family were identified. Three have a seryl residue in the post-tyrosyl position. Two of these are octapeptides that differ only at the N-terminal residue; NRPYSFGL-NH2 and ARPYSFGL-NH2, whilst the third is the heptapeptide derived by N-terminal trimming; RPYSFGL-NH2. Two octapeptides in which X is Ala and Asn were also identified; VERYAFGL-NH2 and LPVYNFGL-NH2. The latter peptide is derived by processing at the internal dibasic site of a putative heneicosapeptide encoded by the DNA. These findings stress the necessity to have putative structures verified at the peptide level. Potent, reversible inhibitory effects on the spontaneous contractile activity of the blowfly rectum were recorded for ARPYSFGL-NH2 (monophasic dose-response curve with an IC50 = 10 fM) and for LPVYNFGL-NH2 (biphasic dose-response curve with IC50 values of approximately 1 fM and 1 nM). It is suggested that regulation of gut motility in insects, rather than an allatostatic function, may represent an ancestral and universal function of the allatostatins. One of the reasons for the large number of members of the Leu-callatostatin family appears to be in the provision of an integrated form of gut motility control, with different peptides controlling specific regions of the gut.

Preliminary characterisation of digestive proteases of the green mirid, Creontiades dilutus (Hemiptera: Miridae).

Protease activities in the secreted saliva, salivary glands and midgut of the green mirid, Creontiades dilutus, were investigated. The saliva and salivary glands had more protease activity than the midgut, but no differences in protease activity levels were detected between male and female mirids, adult mirids and third instar nymphs, or between fed and starved mirids. In the salivary glands, chymotrypsin-like serine proteases predominated, as characterised by inhibitor specificity, basic pH optima, and hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide and N-succinyl-ala-ala-pro-leu p-nitroanilide. The pH optimum of midgut extracts was acidic (pH 4), implying that acidic proteases predominate. However, protease activity was inhibited substantially by both aprotinin and E-64, suggesting the presence of both serine and cysteine proteases in the midgut of the green mirid.